Endothelin-1 may play an important role in the Fontan circulation

Endothelin-1 may play an important role in the Fontan circulation Abstract OBJECTIVES Our goal was to evaluate whether endothlin-1 (ET-1) plays an important role in the Fontan circulation. METHODS Thirteen patients with single-ventricle physiology (Glenn circulation, n = 7; Fontan circulation, n = 6) were evaluated using lung histopathological and immunohistochemical studies and then compared with the normal autopsied controls without congenital heart disease (n = 13). We evaluated the medial thickness of the small pulmonary arteries. For 10 of these patients, quantitative real-time polymerase chain reaction analyses of ET-1, endothelin receptors Type A and Type B, endothelin-converting enzyme-1 and endothelial nitric oxide synthase were performed. RESULTS The medial thickness of the small pulmonary arteries in patients with single-ventricle physiology was greater than that of those in the control group (P = 0.0341). Severe medial hypertrophy of the pulmonary arteries was observed in patients who had poor outcomes. Immunohistochemical studies revealed that the marked expression of ET-1 was observed in the endothelium and media of their pulmonary arteries. In these patients, the messenger RNA expression of ET-1 was also increased. Two patients showed high levels of expression of ETAR and ETBR, although these 2 cases maintain good Fontan circulation. CONCLUSIONS Medial hypertrophy and the overexpression of ET-1 in the pulmonary arteries were observed in some patients in whom the Fontan circulation failed. Our data suggest that ET-1 may play an important role in maintaining the Fontan circulation. Endothelin, Fontan circulation, Pulmonary hypertension INTRODUCTION Since Fontan and Baudet [1] first described their procedure for the correction of tricuspid atresia in 1971, patients with single-ventricle (SV) physiology have been treated with the Fontan procedure. Several modifications of this operation and advances in postoperative management have improved the surgical results, despite the application of the Fontan approach to patients with complex cardiac anomalies [2, 3]. The outcome of the Fontan procedure is highly dependent on several risk factors, one of which is pulmonary vascular resistance (PVR) [4, 5]. In the absence of a pulmonary ventricle, even a slight increase in PVR can result in failure of the Fontan circulation. It is well known that the Fontan procedure failed in some patients despite fulfilment of the usual haemodynamic criterion [6]. Several studies revealed that an increased medial thickness of the small pulmonary arteries was observed in patients whose pulmonary arterial pressure (PAP) and PVR were normal [7, 8]. Some authors suggested that endothelial dysfunction could be involved in these patients. Lévy et al. [9] revealed that endothelial nitric oxide synthase (eNOS) was markedly overexpressed in patients whose small pulmonary arteries displayed muscle extension with an increased wall thickness. Ishida et al. [10] also found significant medial hypertrophy with proliferation of vascular smooth muscle cells and the overexpression of endothelin-1 (ET-1) in the intra-acinar pulmonary arteries of the autopsy lung tissues of failed Fontan patients. ET-1 is one of the potent endogenous vasoconstrictors. We recently showed that bosentan, an oral endothelin receptor antagonist, reduced the PAP and PVR in patients with SV physiology who were initially unable to undergo a bidirectional Glenn (BDG) or the Fontan operation because of high PAP and PVR [11]. All patients had improved clinical symptoms and underwent a successful Fontan procedure. Thus, we hypothesized that the endothelin system may play an important role in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling in patients with SV physiology. We evaluated lung specimens taken from patients who underwent a BDG or the Fontan operation using histomorphometrical, immunohistochemical and quantitative real-time polymerase chain reaction (RT-PCR) analyses. MATERIALS AND METHODS Study population Between 2012 and 2015, we performed histopathological analyses on biopsy specimens of lung tissue from 13 patients with SV physiology aged 1–27 years (mean 5.8 ± 7.0 years). Open lung biopsies, mostly of the right upper lobe, were initially performed during the operation. The Fontan procedure was performed via a median sternotomy and standard cardiopulmonary bypass with mild hypothermia. As an extracardiac conduit, an expanded polytetrafluoroethylene conduit was used in all patients. Biopsy specimens from all 13 patients operated on during this period were analysed. Eleven patients underwent cardiac catheterization before having Fontan operations or comorbidity operations. The patient characteristics are summarized in Tables 1–3 and Supplementary Material, Table S1. A lung biopsy was performed in 7 patients with Glenn circulation (Cases 1–7) and in 6 patients with Fontan circulation (Cases 8–13). One patient (Case 1) required takedown of the Fontan circulation because of conduit obstruction 1 month after the Fontan operation. Two patients were unable to undergo Fontan operation because of severe ventricular dysfunction (Case 2) and pulmonary hypertension due to numerous collateral arteries (Case 3). One patient with Down syndrome (Case 4) died of congestive heart failure 1 month after the Fontan operation. Two patients suffered from phrenic nerve paralysis, although they did not need ventilatory support. One patient (Case 9) had a good Fontan physiology for 2 years, but we tried to further improve it by plication of the diaphragm. The other patient (Case 13) had severe hypercapnia for 1 month after the Fontan operation, which led us to perform plication of the diaphragm. We obtained controlled lung autopsy specimens from 13 patients aged 4 months to 27 years (mean 5.4 ± 7.2 years) without congenital heart disease (Supplementary Material, Table S2). Table 1: Clinical characteristics of the patients Case  Gender  Age at lung biopsy (years)  Diagnosis  Operations  Glenn circulation   1  M  2  SV, PA, pulmonary CoA, apicocaval juxtaposition  TCPC   2  F  1  HLHS variant  CRT   3  M  6  SV, PS, TAPVC, CAVV, asplenia  CAVV replacement   4  M  5  Fallot, AVSD, multiple VSD, Down’s syndrome  TCPC   5  F  3  SV, PA, TAPVC, pulmonary CoA, asplenia  TCPC   6  F  3  DORV, MS (hypo LV)  TCPC   7  F  1  TGA, PS, multiple VSD  TCPC  Fontan circulation   8  F  27  TA  TCPC conversion   9  M  4  TA  Plication of diaphragm   10  F  3  SV, PS, TAPVC, apicocaval juxtaposition, asplenia  Release of left PVO   11  F  10  SV, PS, CAVV, TAPVC, asplenia  Redo CAVV replacement   12  M  12  SV, PS, CAVV, TAPVC, asplenia  Third CAVV replacement   13  M  2  CoA, false Taussig-Bing anomaly, multiple VSD  Plication of diaphragm  Case  Gender  Age at lung biopsy (years)  Diagnosis  Operations  Glenn circulation   1  M  2  SV, PA, pulmonary CoA, apicocaval juxtaposition  TCPC   2  F  1  HLHS variant  CRT   3  M  6  SV, PS, TAPVC, CAVV, asplenia  CAVV replacement   4  M  5  Fallot, AVSD, multiple VSD, Down’s syndrome  TCPC   5  F  3  SV, PA, TAPVC, pulmonary CoA, asplenia  TCPC   6  F  3  DORV, MS (hypo LV)  TCPC   7  F  1  TGA, PS, multiple VSD  TCPC  Fontan circulation   8  F  27  TA  TCPC conversion   9  M  4  TA  Plication of diaphragm   10  F  3  SV, PS, TAPVC, apicocaval juxtaposition, asplenia  Release of left PVO   11  F  10  SV, PS, CAVV, TAPVC, asplenia  Redo CAVV replacement   12  M  12  SV, PS, CAVV, TAPVC, asplenia  Third CAVV replacement   13  M  2  CoA, false Taussig-Bing anomaly, multiple VSD  Plication of diaphragm  AVSD: atrioventricular septal defect; CAVV: common atrioventricular valve; CoA: coarctation; CRT: cardiac resynchronization therapy; DORV: double-outlet right ventricle; F: female; HLHS: hypoplastic left heart syndrome; LV: left ventricle; M: male; MS: mitral stenosis; PA: pulmonary atresia; PS: pulmonary stenosis; PVO: pulmonary venous obstruction; SV: single ventricle; TA: tricuspid atresia; TAPVC: total anomalous of pulmonary venous connection; TCPC: total cavopulmonary connection; TGA: transposition of the great arteries; VSD: ventricular septal defect. Table 2: Clinical and pathological patient data Case  CVP (mmHg)  Mean PAP (mmHg)  PCWP (mmHg)  PVR (units × m2)  Bosentan  DR = 100μm (μm)  RT-PCR ET-1  RT-PCR eNOS  1  7  6  5  1.7  On  23.9  1.26  1.6  2  13  13  10  2.4  On  12.7  0.98  0.9  3  24  24  20  2.5  Off  19.0  1.72  1.3  4  13  12  13  1.5  On  19.9  1.87  2.3  5  13  12  8  1.3  On  4.9      6  17  17    2.7  On  5.6  1.03  2.4  7  16  15    2.7  Off  13.0  0.82  2.2  8          Off  8.4      9  8  7  6  1.3  Off  5.8  0.70  3.8  10          Off  22.1  0.52  1.3  11  16  16  12  1.5  Off  14.2  1.55  2.5  12  22  22  4  0.5  Off  5.4  1.45  1.7  13  12  16  12  1.2  On  9.3      Case  CVP (mmHg)  Mean PAP (mmHg)  PCWP (mmHg)  PVR (units × m2)  Bosentan  DR = 100μm (μm)  RT-PCR ET-1  RT-PCR eNOS  1  7  6  5  1.7  On  23.9  1.26  1.6  2  13  13  10  2.4  On  12.7  0.98  0.9  3  24  24  20  2.5  Off  19.0  1.72  1.3  4  13  12  13  1.5  On  19.9  1.87  2.3  5  13  12  8  1.3  On  4.9      6  17  17    2.7  On  5.6  1.03  2.4  7  16  15    2.7  Off  13.0  0.82  2.2  8          Off  8.4      9  8  7  6  1.3  Off  5.8  0.70  3.8  10          Off  22.1  0.52  1.3  11  16  16  12  1.5  Off  14.2  1.55  2.5  12  22  22  4  0.5  Off  5.4  1.45  1.7  13  12  16  12  1.2  On  9.3      CVP: central venous pressure; eNOS: endothelial nitric oxide synthase; ET-1: endothelin-1; PAP: pulmonary artery pressure; PCWP: pulmonary capillary wedge pressure; PVR: pulmonary vascular resistance; RT-PCR: real-time polymerase chain reaction. Table 3: Patient clinical data Case  BSA (m2)  Pulse oximetry (%)  Qp/Qs  PA index  CAVVR  NT pro-BNP (ng/ml)  1  0.54  88  0.82  159  None  501  2  0.39  84  0.40  80  None  17 269  3  0.60  79  1.37  188  Severe  4417  4  0.61  87  0.81  325  None  471  5  0.56  87  0.61    None  45  6  0.47  88  0.85  280  None  424  7  0.49  88  0.53  180  None  319  8  1.27  99      Mild    9  0.58  95  0.81  386  None  55  10  0.52  99      None  486  11  0.99  96  1.00  177  None  110  12  0.90  95  1.00  79  None  415  13  0.50  99  1.17  293  None  695  Case  BSA (m2)  Pulse oximetry (%)  Qp/Qs  PA index  CAVVR  NT pro-BNP (ng/ml)  1  0.54  88  0.82  159  None  501  2  0.39  84  0.40  80  None  17 269  3  0.60  79  1.37  188  Severe  4417  4  0.61  87  0.81  325  None  471  5  0.56  87  0.61    None  45  6  0.47  88  0.85  280  None  424  7  0.49  88  0.53  180  None  319  8  1.27  99      Mild    9  0.58  95  0.81  386  None  55  10  0.52  99      None  486  11  0.99  96  1.00  177  None  110  12  0.90  95  1.00  79  None  415  13  0.50  99  1.17  293  None  695  BSA: body surface area; CAVVR: common atrioventricular regurgitation; NT pro-BNP: N-terminal pro-brain natriuretic peptide; PA: pulmonary arterial. Histopathological study of lung specimens The lung specimens were fixed in 4% paraformaldehyde phosphate-buffered solution for 24 h. Serial paraffin-embedded 5-μm-thick sections were cut, deparaffinized in xylene and rehydrated in graded alcohol. The sections were stained with haematoxylin–eosin, Masson’s trichrome and Elastica van Gieson. The pulmonary arteries in a section were scanned using a microscope (Olympus B50 and DP73, Tokyo, Japan). The medial thickness of the pulmonary arteries was calculated using the methods described by Yamaki and Tezuka [12] and Maeda et al. [13] as follows (Supplementary Material, Fig. S1). This method uses standardized media without the influence of pulmonary arterial contraction. The surface area (S) of the medial cross-section and the length (L) of the internal elastic membrane were measured using the ImageJ software program (http://www.rsb.info.nih.gov/ij/). The medial thickness (D) and the radius (R), the distance from the centre of the pulmonary artery to the mid-point of the media, were calculated from the following formulae:   D = [(L2 + 4πS)1/2−L]/2π, R = S/[(L2 + 4πS)1/2−L]. Figure 1: View largeDownload slide Morphological analyses of the degree of media of the pulmonary artery. The relationship between DR = 100 and ventricle physiology. There was a significant difference between the 2 groups (P = 0.0341). Error bars represent 95% confidence intervals. Concentrations are expressed in micrometres. SV: single ventricle. Figure 1: View largeDownload slide Morphological analyses of the degree of media of the pulmonary artery. The relationship between DR = 100 and ventricle physiology. There was a significant difference between the 2 groups (P = 0.0341). Error bars represent 95% confidence intervals. Concentrations are expressed in micrometres. SV: single ventricle. The values of R and D were plotted on a logarithmic coordinate system. The mean number of calculated pulmonary arteries was 32.7 (17–78). Linear regression analyses of R and D were performed. The D value at R = 100 μm (DR=100μm) was computed to compare with normal controls as the degree of pulmonary arterial hypertrophy. Immunohistochemical and immunofluorescence studies Sections were immunostained with anti-ET-1(Abnova, H00001906-M01, Taiwan) to evaluate proliferation of the media. The details of the staining technique are shown in Supplementary Document S1. Slides were examined using the ZEISS LSM 700 with an Airyscan microscope (Carl Zeiss, Japan). Quantitative real-time polymerase chain reaction studies All except 3 biopsy specimens (Cases 5, 8 and 13) were available for RT-PCR analyses. Total RNA was prepared from lung tissues, and complementary DNA was synthesized from total RNA using reverse transcriptase (TaKaRa Bio, Kusatsu, Japan). Quantitative RT-PCR was performed with the LightCycler96 system (Roche Diagnostics, Basel, Switzerland) using the following PCR conditions: 40 cycles 95 °C for 10 s, 52 °C for 10 s and 72 °C for 10 s. For ETBR, the PCR conditions were 40 cycles 95 °C for 20 s, 55 °C for 20 s and 72 °C for 20 s. The oligonucleotides used for PCR amplification are shown in Supplementary Document S2. Statistical analyses All values are presented as the mean ± standard deviation. When the data followed a normal distribution, comparisons between 2 groups were performed using the Student’s t-test. When the data did not follow a normal distribution, comparisons between 2 groups were performed using the Wilcoxon rank-sum test. All tests were 2-tailed, and statistical significance was assessed at the 5% α level. Research ethics board approval The research ethics committee of the University of Toyama approved this study. Written informed consent was obtained from the patients’ parents after the aim of these studies was explained to them. RESULTS Histopathological studies (DR=100μm) of each case are presented in Table 2. The mean (DR=100μm) values in patients with SV physiology were significantly greater than those in the control group (P = 0.0341, 12.6 ± 6.8 vs. 8.4 ± 1.0μm) (Fig. 1). Four patients (Cases 1, 3, 4 and 10) had severe medial hypertrophy of the pulmonary arteries (Fig. 2A). Their (DR=100μm)values amounted to nearly 20 μm. Three of these 4 patients (Cases 1, 3 and 4) failed or were not suitable for the Fontan operation, as described previously. The other patient (Case 10), who had asplenia syndrome, required release of a left pulmonary venous obstruction 10 months after the Fontan operation. A lung specimen was obtained from the patient’s left lung. The patient is doing well 2 years after the redo operation. Eight patients (Cases 5, 6, 7, 8, 9, 11, 12 and 13) with no or mild medial hypertrophy of the pulmonary arteries maintain good Fontan circulation (Fig 2B, 3C). There were no significant correlations between the (DR=100μm) values and any haemodynamic parameters. Figure 2: View largeDownload slide Histopathological findings of lung specimens. (A) Elastica van Gieson staining of the pulmonary artery in a patient who did not undergo a successful Fontan operation shows both severe intimal and medial hypertrophy (Case 1). (B) Elastica van Gieson staining of pulmonary arteries in a patient with a satisfactory clinical course showed slightly hypertrophic media (Case 7). (C) Masson’s trichrome staining of the pulmonary arteries in a normal control shows thin media. Figure 2: View largeDownload slide Histopathological findings of lung specimens. (A) Elastica van Gieson staining of the pulmonary artery in a patient who did not undergo a successful Fontan operation shows both severe intimal and medial hypertrophy (Case 1). (B) Elastica van Gieson staining of pulmonary arteries in a patient with a satisfactory clinical course showed slightly hypertrophic media (Case 7). (C) Masson’s trichrome staining of the pulmonary arteries in a normal control shows thin media. Figure 3: View largeDownload slide Endothelin-1 (green) and DAPI (blue) immunofluorescence staining of pulmonary arteries. (A) In a patient who did not undergo a successful Fontan operation, both the endothelium and media were strongly stained (Case 1). White arrowheads indicate the division between the media and the intima. (B) In a patient with a satisfactory clinical course, ET-1 was observed in the endothelium (Case 7). (C) In the normal control, the media and endothelium were barely stained. Figure 3: View largeDownload slide Endothelin-1 (green) and DAPI (blue) immunofluorescence staining of pulmonary arteries. (A) In a patient who did not undergo a successful Fontan operation, both the endothelium and media were strongly stained (Case 1). White arrowheads indicate the division between the media and the intima. (B) In a patient with a satisfactory clinical course, ET-1 was observed in the endothelium (Case 7). (C) In the normal control, the media and endothelium were barely stained. Immunohistochemical and immunofluorescence studies Three patients (Cases 1, 3, and 4) with severe medial hypertrophy of the pulmonary arteries showed a marked expression of ET-1 immunoreactivity in the endothelium and media of their pulmonary arteries (Fig. 3A). ET-1 immunoreactivity was mainly observed in the endothelium of pulmonary arteries in other patients and in those in the control group (Fig. 3B and C). Quantitative real-time polymerase chain reaction studies The results of RT-PCR analyses are shown in Table 2 and Fig. 4. Three patients who failed or were not suitable for the Fontan operation (Cases 1, 3 and 4) showed high levels of ET-1 expression, corresponding to the results of immunohistochemical and immunofluorescence studies. Two patients (Cases 11 and 12) showed high levels of ET-1. These 2 patients underwent redo common atrioventricular valve replacement for relative prosthetic valve obstruction because of somatic growth. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR expression, although these 2 cases maintain good Fontan circulation. There were no particular findings regarding the expression levels of ECE-1 and eNOS. There were no significant correlations between the duration of the Fontan circulation and the levels of ET-1, eNOS and ET receptors. Figure 4: View largeDownload slide Real-time polymerase chain reaction analyses of lung biopsies. (A) ET-1, (B) ETAR, (C) ETBR, (D) ECE-1 and (E) eNOS. Five patients (Cases 1, 3, 4, 11 and 12) showed high levels of ET-1. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR. Figure 4: View largeDownload slide Real-time polymerase chain reaction analyses of lung biopsies. (A) ET-1, (B) ETAR, (C) ETBR, (D) ECE-1 and (E) eNOS. Five patients (Cases 1, 3, 4, 11 and 12) showed high levels of ET-1. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR. DISCUSSION ET-1, a 21-amino acid peptide discovered in 1988 by Yanagisawa et al. [14], is one of the most potent endogenous vasoconstrictors and has mitogenic properties [15, 16]. ET-1 is strongly expressed in the lung, with ET-1 messenger RNA levels being at least 5-fold greater than in any other organ [17]. ET-1 in the pulmonary circulation can induce intense and protracted vasoconstriction of the pulmonary arteries and veins [18]. In addition to its effects on the pulmonary vascular tone, ET-1 also has a weak mitogenic effect on pulmonary vascular smooth muscle cells and stimulates matrix production by the vessel wall [19]. Although Hiramatsu et al. [5] demonstrated that ET-1 elevated PVR in patients with Fontan circulation, there were limited data about the relationship between ET-1 and the Fontan circulation. PVR is one of the most critical factors for maintaining the Fontan circulation and is dependent on various vasoactive factors, such as nitric oxide and prostacyclins. The pathophysiological role of ET-1 in PVR, in particular the potential involvement of ET-1 in patients with Fontan circulation, remains controversial. The aim of our study was thus to evaluate whether ET-1 plays an important role in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling in patients with SV physiology. We showed that patients who were unable to undergo a successful Fontan operation had both pulmonary arterial hypertrophy and the overexpression of ET-1 in the media of the pulmonary arteries using lung specimens. Maeda et al. [13] revealed that severe medial hypertrophy of the pulmonary arteries was correlated with a poor outcome of the Fontan operation. Indeed, our 3 patients (Cases 1, 3 and 4) with severe medial hypertrophy of the pulmonary arteries had poor outcomes (Fig. 2A). ET-1 immunoreactivity in these patients was observed in the endothelium and media (Fig. 3A). Thus, ET-1 may be associated with medial hypertrophy of the pulmonary arteries in the Fontan circulation. Conversely, patients without medial hypertrophy had good clinical courses (Fig. 2B), and ET-1 immunoreactivity was not observed in the media (Fig. 3B). Ishida et al. [10] also reported that the pulmonary arteries of the patients with failing Fontan circulation exhibited significant medial hypertrophy with proliferation of vascular smooth muscle cells and the expression of ET-1. ET-1 may play a role in medial hypertrophy of the pulmonary arteries in patients with Fontan circulation. We performed RT-PCR analyses to evaluate the quantity of the expression of ET-1. We revealed that the expression levels of ET-1 were increased in 5 patients (Cases 1, 3, 4, 11 and 12). Two of these patients (Cases 1 and 3) had poor early outcomes after the Fontan operation, despite normal PAP and PVR. One patient (Case 3) was not considered a candidate for Fontan completion because of pulmonary hypertension due to severe common atrioventricular regurgitation and numerous collateral arteries. These 3 patients had severe medial hypertrophy of the pulmonary arteries and marked expression of ET-1 immunoreactivity in the histopathological studies. However, vascular remodelling might be induced by ET-1, ET-1 would be one of the remodelling factors (cyanosis, common atrioventricular valve and pulmonary venous obstruction). Three of these 5 patients (Cases 3, 11 and 12) might have had an additional potential effect of post-capillary pulmonary hypertension. In our previous study of bosentan therapy, 2 of 8 patients showed an improvement in post-capillary pulmonary hypertension and underwent the Fontan operation successfully [11]. Bosentan therapy might be tried to treat post-capillary pulmonary hypertension, although the post-capillary pulmonary hypertension should be treated by surgical intervention if possible. Pulmonary arterial hypertrophy and the overexpression of ET-1 might be warnings of failing Fontan circulation, even though PAP and PVR were within normal limits. Because ET-1 also has a weak mitogenic effect on pulmonary vascular smooth muscle cells [19], this pathological status might disturb the Fontan circulation. The expressions of both ETAR and ETBR increased in 2 patients (Fig. 4B and C). These 2 patients (Cases 7 and 9) had satisfactory clinical courses. Ishida et al. [10] mentioned that the expression levels of ETAR and ETBR were significantly elevated in the pulmonary arteries of patients whose Fontan circulation failed. However, they did not study the endothelin system in patients who had good clinical courses after the Fontan operation. Recent pharmacological studies showed that ETAR and ETBR may form homodimers and/or heterodimers under pathological conditions [20, 21]. The Fontan circulation itself might be one of the pathological conditions. The expressions of ETAR and ETBR could increase in patients with Fontan circulation even if their circulation is good. Moreover, the endothelin systems might be complicated in patients with SV physiology. In the Fontan circulation, a non-pulsatile blood flow could create shear stress on the endothelium of the pulmonary system [22, 23]. As described previously, we reported that bosentan therapy reduced the PAP and PVR in patients who were unable to undergo a BDG or the Fontan operation because of high PAP and PVR [11]. Bosentan is an orally active, dual (ETAR and ETBR) endothelin receptor antagonist that can block both the detrimental effects of ET mediated via ETAR and the beneficial actions mediated via ETBR [24]. This study showed that ET-1 may play a role in promoting medial hypertrophy of the pulmonary arteries in patients with Fontan circulation. Our findings might suggest the validity of endothelin receptor antagonist therapy for patients with Fontan circulation. Limitations There are some limitations associated with this study. First, our study population was small. In the future, we should divide patients according to the type (Fontan or Glenn) of circulation. Second, we conducted immunohistochemical studies, which are not quantitative. In the RT-PCR studies, the lack of a comparison group of patients with BV physiology prevented us from presenting definitive evidence. Taking into consideration the multiple factors that may affect SV physiology, a group of patients with BV physiology would have been more suitable as a control group; however, such a group would have been difficult to incorporate into the study because few patients undergo lung biopsy without illness in infancy. CONCLUSIONS Severe medial hypertrophy of the pulmonary arteries was observed in patients who had poor outcomes. Marked expression of ET-1 was also observed in the endothelium and media of their pulmonary arteries. These pathological conditions may have some influence on the Fontan circulation. Thus, we concluded that there is an association between ET-1 and SV physiology in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling. SUPPLEMENTARY MATERIAL Supplementary material is available at ICVTS online. ACKNOWLEDGMENTS The authors wish to thank Hitoshi Moriuchi, Hatta Hideki, Yukiko Hata, Kazuhiro Watanabe, Sayaka Ozawa, Keijirou Ibuki, Kazuyoshi Saito and Hideyuki Nakaoka for assistance. Conflict of interest: none declared. REFERENCES 1 Fontan F, Baudet E. Surgical repair of tricuspid atresia. Thorax  1971; 26: 240– 8. http://dx.doi.org/10.1136/thx.26.3.240 Google Scholar CrossRef Search ADS PubMed  2 Jacobs JP, O'Brien SM, Chai PJ, Morell VO, Lindberg HL, Quintessenza JA. Management of 239 patients with hypoplastic left heart syndrome and related malformations from 1993 to 2007. Ann Thorac Surg  2008; 85: 1691– 7. Google Scholar CrossRef Search ADS PubMed  3 Yoshimura N, Yamaguchi M, Oshima Y, Oka S, Ootaki Y, Hasegawa T et al.   Suppression of the secretion of atrial and brain natriuretic peptide after total cavopulmonary connection. J Thorac Cardiovasc Surg  2000; 120: 764– 9. 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© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.
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Abstract

Abstract OBJECTIVES Our goal was to evaluate whether endothlin-1 (ET-1) plays an important role in the Fontan circulation. METHODS Thirteen patients with single-ventricle physiology (Glenn circulation, n = 7; Fontan circulation, n = 6) were evaluated using lung histopathological and immunohistochemical studies and then compared with the normal autopsied controls without congenital heart disease (n = 13). We evaluated the medial thickness of the small pulmonary arteries. For 10 of these patients, quantitative real-time polymerase chain reaction analyses of ET-1, endothelin receptors Type A and Type B, endothelin-converting enzyme-1 and endothelial nitric oxide synthase were performed. RESULTS The medial thickness of the small pulmonary arteries in patients with single-ventricle physiology was greater than that of those in the control group (P = 0.0341). Severe medial hypertrophy of the pulmonary arteries was observed in patients who had poor outcomes. Immunohistochemical studies revealed that the marked expression of ET-1 was observed in the endothelium and media of their pulmonary arteries. In these patients, the messenger RNA expression of ET-1 was also increased. Two patients showed high levels of expression of ETAR and ETBR, although these 2 cases maintain good Fontan circulation. CONCLUSIONS Medial hypertrophy and the overexpression of ET-1 in the pulmonary arteries were observed in some patients in whom the Fontan circulation failed. Our data suggest that ET-1 may play an important role in maintaining the Fontan circulation. Endothelin, Fontan circulation, Pulmonary hypertension INTRODUCTION Since Fontan and Baudet [1] first described their procedure for the correction of tricuspid atresia in 1971, patients with single-ventricle (SV) physiology have been treated with the Fontan procedure. Several modifications of this operation and advances in postoperative management have improved the surgical results, despite the application of the Fontan approach to patients with complex cardiac anomalies [2, 3]. The outcome of the Fontan procedure is highly dependent on several risk factors, one of which is pulmonary vascular resistance (PVR) [4, 5]. In the absence of a pulmonary ventricle, even a slight increase in PVR can result in failure of the Fontan circulation. It is well known that the Fontan procedure failed in some patients despite fulfilment of the usual haemodynamic criterion [6]. Several studies revealed that an increased medial thickness of the small pulmonary arteries was observed in patients whose pulmonary arterial pressure (PAP) and PVR were normal [7, 8]. Some authors suggested that endothelial dysfunction could be involved in these patients. Lévy et al. [9] revealed that endothelial nitric oxide synthase (eNOS) was markedly overexpressed in patients whose small pulmonary arteries displayed muscle extension with an increased wall thickness. Ishida et al. [10] also found significant medial hypertrophy with proliferation of vascular smooth muscle cells and the overexpression of endothelin-1 (ET-1) in the intra-acinar pulmonary arteries of the autopsy lung tissues of failed Fontan patients. ET-1 is one of the potent endogenous vasoconstrictors. We recently showed that bosentan, an oral endothelin receptor antagonist, reduced the PAP and PVR in patients with SV physiology who were initially unable to undergo a bidirectional Glenn (BDG) or the Fontan operation because of high PAP and PVR [11]. All patients had improved clinical symptoms and underwent a successful Fontan procedure. Thus, we hypothesized that the endothelin system may play an important role in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling in patients with SV physiology. We evaluated lung specimens taken from patients who underwent a BDG or the Fontan operation using histomorphometrical, immunohistochemical and quantitative real-time polymerase chain reaction (RT-PCR) analyses. MATERIALS AND METHODS Study population Between 2012 and 2015, we performed histopathological analyses on biopsy specimens of lung tissue from 13 patients with SV physiology aged 1–27 years (mean 5.8 ± 7.0 years). Open lung biopsies, mostly of the right upper lobe, were initially performed during the operation. The Fontan procedure was performed via a median sternotomy and standard cardiopulmonary bypass with mild hypothermia. As an extracardiac conduit, an expanded polytetrafluoroethylene conduit was used in all patients. Biopsy specimens from all 13 patients operated on during this period were analysed. Eleven patients underwent cardiac catheterization before having Fontan operations or comorbidity operations. The patient characteristics are summarized in Tables 1–3 and Supplementary Material, Table S1. A lung biopsy was performed in 7 patients with Glenn circulation (Cases 1–7) and in 6 patients with Fontan circulation (Cases 8–13). One patient (Case 1) required takedown of the Fontan circulation because of conduit obstruction 1 month after the Fontan operation. Two patients were unable to undergo Fontan operation because of severe ventricular dysfunction (Case 2) and pulmonary hypertension due to numerous collateral arteries (Case 3). One patient with Down syndrome (Case 4) died of congestive heart failure 1 month after the Fontan operation. Two patients suffered from phrenic nerve paralysis, although they did not need ventilatory support. One patient (Case 9) had a good Fontan physiology for 2 years, but we tried to further improve it by plication of the diaphragm. The other patient (Case 13) had severe hypercapnia for 1 month after the Fontan operation, which led us to perform plication of the diaphragm. We obtained controlled lung autopsy specimens from 13 patients aged 4 months to 27 years (mean 5.4 ± 7.2 years) without congenital heart disease (Supplementary Material, Table S2). Table 1: Clinical characteristics of the patients Case  Gender  Age at lung biopsy (years)  Diagnosis  Operations  Glenn circulation   1  M  2  SV, PA, pulmonary CoA, apicocaval juxtaposition  TCPC   2  F  1  HLHS variant  CRT   3  M  6  SV, PS, TAPVC, CAVV, asplenia  CAVV replacement   4  M  5  Fallot, AVSD, multiple VSD, Down’s syndrome  TCPC   5  F  3  SV, PA, TAPVC, pulmonary CoA, asplenia  TCPC   6  F  3  DORV, MS (hypo LV)  TCPC   7  F  1  TGA, PS, multiple VSD  TCPC  Fontan circulation   8  F  27  TA  TCPC conversion   9  M  4  TA  Plication of diaphragm   10  F  3  SV, PS, TAPVC, apicocaval juxtaposition, asplenia  Release of left PVO   11  F  10  SV, PS, CAVV, TAPVC, asplenia  Redo CAVV replacement   12  M  12  SV, PS, CAVV, TAPVC, asplenia  Third CAVV replacement   13  M  2  CoA, false Taussig-Bing anomaly, multiple VSD  Plication of diaphragm  Case  Gender  Age at lung biopsy (years)  Diagnosis  Operations  Glenn circulation   1  M  2  SV, PA, pulmonary CoA, apicocaval juxtaposition  TCPC   2  F  1  HLHS variant  CRT   3  M  6  SV, PS, TAPVC, CAVV, asplenia  CAVV replacement   4  M  5  Fallot, AVSD, multiple VSD, Down’s syndrome  TCPC   5  F  3  SV, PA, TAPVC, pulmonary CoA, asplenia  TCPC   6  F  3  DORV, MS (hypo LV)  TCPC   7  F  1  TGA, PS, multiple VSD  TCPC  Fontan circulation   8  F  27  TA  TCPC conversion   9  M  4  TA  Plication of diaphragm   10  F  3  SV, PS, TAPVC, apicocaval juxtaposition, asplenia  Release of left PVO   11  F  10  SV, PS, CAVV, TAPVC, asplenia  Redo CAVV replacement   12  M  12  SV, PS, CAVV, TAPVC, asplenia  Third CAVV replacement   13  M  2  CoA, false Taussig-Bing anomaly, multiple VSD  Plication of diaphragm  AVSD: atrioventricular septal defect; CAVV: common atrioventricular valve; CoA: coarctation; CRT: cardiac resynchronization therapy; DORV: double-outlet right ventricle; F: female; HLHS: hypoplastic left heart syndrome; LV: left ventricle; M: male; MS: mitral stenosis; PA: pulmonary atresia; PS: pulmonary stenosis; PVO: pulmonary venous obstruction; SV: single ventricle; TA: tricuspid atresia; TAPVC: total anomalous of pulmonary venous connection; TCPC: total cavopulmonary connection; TGA: transposition of the great arteries; VSD: ventricular septal defect. Table 2: Clinical and pathological patient data Case  CVP (mmHg)  Mean PAP (mmHg)  PCWP (mmHg)  PVR (units × m2)  Bosentan  DR = 100μm (μm)  RT-PCR ET-1  RT-PCR eNOS  1  7  6  5  1.7  On  23.9  1.26  1.6  2  13  13  10  2.4  On  12.7  0.98  0.9  3  24  24  20  2.5  Off  19.0  1.72  1.3  4  13  12  13  1.5  On  19.9  1.87  2.3  5  13  12  8  1.3  On  4.9      6  17  17    2.7  On  5.6  1.03  2.4  7  16  15    2.7  Off  13.0  0.82  2.2  8          Off  8.4      9  8  7  6  1.3  Off  5.8  0.70  3.8  10          Off  22.1  0.52  1.3  11  16  16  12  1.5  Off  14.2  1.55  2.5  12  22  22  4  0.5  Off  5.4  1.45  1.7  13  12  16  12  1.2  On  9.3      Case  CVP (mmHg)  Mean PAP (mmHg)  PCWP (mmHg)  PVR (units × m2)  Bosentan  DR = 100μm (μm)  RT-PCR ET-1  RT-PCR eNOS  1  7  6  5  1.7  On  23.9  1.26  1.6  2  13  13  10  2.4  On  12.7  0.98  0.9  3  24  24  20  2.5  Off  19.0  1.72  1.3  4  13  12  13  1.5  On  19.9  1.87  2.3  5  13  12  8  1.3  On  4.9      6  17  17    2.7  On  5.6  1.03  2.4  7  16  15    2.7  Off  13.0  0.82  2.2  8          Off  8.4      9  8  7  6  1.3  Off  5.8  0.70  3.8  10          Off  22.1  0.52  1.3  11  16  16  12  1.5  Off  14.2  1.55  2.5  12  22  22  4  0.5  Off  5.4  1.45  1.7  13  12  16  12  1.2  On  9.3      CVP: central venous pressure; eNOS: endothelial nitric oxide synthase; ET-1: endothelin-1; PAP: pulmonary artery pressure; PCWP: pulmonary capillary wedge pressure; PVR: pulmonary vascular resistance; RT-PCR: real-time polymerase chain reaction. Table 3: Patient clinical data Case  BSA (m2)  Pulse oximetry (%)  Qp/Qs  PA index  CAVVR  NT pro-BNP (ng/ml)  1  0.54  88  0.82  159  None  501  2  0.39  84  0.40  80  None  17 269  3  0.60  79  1.37  188  Severe  4417  4  0.61  87  0.81  325  None  471  5  0.56  87  0.61    None  45  6  0.47  88  0.85  280  None  424  7  0.49  88  0.53  180  None  319  8  1.27  99      Mild    9  0.58  95  0.81  386  None  55  10  0.52  99      None  486  11  0.99  96  1.00  177  None  110  12  0.90  95  1.00  79  None  415  13  0.50  99  1.17  293  None  695  Case  BSA (m2)  Pulse oximetry (%)  Qp/Qs  PA index  CAVVR  NT pro-BNP (ng/ml)  1  0.54  88  0.82  159  None  501  2  0.39  84  0.40  80  None  17 269  3  0.60  79  1.37  188  Severe  4417  4  0.61  87  0.81  325  None  471  5  0.56  87  0.61    None  45  6  0.47  88  0.85  280  None  424  7  0.49  88  0.53  180  None  319  8  1.27  99      Mild    9  0.58  95  0.81  386  None  55  10  0.52  99      None  486  11  0.99  96  1.00  177  None  110  12  0.90  95  1.00  79  None  415  13  0.50  99  1.17  293  None  695  BSA: body surface area; CAVVR: common atrioventricular regurgitation; NT pro-BNP: N-terminal pro-brain natriuretic peptide; PA: pulmonary arterial. Histopathological study of lung specimens The lung specimens were fixed in 4% paraformaldehyde phosphate-buffered solution for 24 h. Serial paraffin-embedded 5-μm-thick sections were cut, deparaffinized in xylene and rehydrated in graded alcohol. The sections were stained with haematoxylin–eosin, Masson’s trichrome and Elastica van Gieson. The pulmonary arteries in a section were scanned using a microscope (Olympus B50 and DP73, Tokyo, Japan). The medial thickness of the pulmonary arteries was calculated using the methods described by Yamaki and Tezuka [12] and Maeda et al. [13] as follows (Supplementary Material, Fig. S1). This method uses standardized media without the influence of pulmonary arterial contraction. The surface area (S) of the medial cross-section and the length (L) of the internal elastic membrane were measured using the ImageJ software program (http://www.rsb.info.nih.gov/ij/). The medial thickness (D) and the radius (R), the distance from the centre of the pulmonary artery to the mid-point of the media, were calculated from the following formulae:   D = [(L2 + 4πS)1/2−L]/2π, R = S/[(L2 + 4πS)1/2−L]. Figure 1: View largeDownload slide Morphological analyses of the degree of media of the pulmonary artery. The relationship between DR = 100 and ventricle physiology. There was a significant difference between the 2 groups (P = 0.0341). Error bars represent 95% confidence intervals. Concentrations are expressed in micrometres. SV: single ventricle. Figure 1: View largeDownload slide Morphological analyses of the degree of media of the pulmonary artery. The relationship between DR = 100 and ventricle physiology. There was a significant difference between the 2 groups (P = 0.0341). Error bars represent 95% confidence intervals. Concentrations are expressed in micrometres. SV: single ventricle. The values of R and D were plotted on a logarithmic coordinate system. The mean number of calculated pulmonary arteries was 32.7 (17–78). Linear regression analyses of R and D were performed. The D value at R = 100 μm (DR=100μm) was computed to compare with normal controls as the degree of pulmonary arterial hypertrophy. Immunohistochemical and immunofluorescence studies Sections were immunostained with anti-ET-1(Abnova, H00001906-M01, Taiwan) to evaluate proliferation of the media. The details of the staining technique are shown in Supplementary Document S1. Slides were examined using the ZEISS LSM 700 with an Airyscan microscope (Carl Zeiss, Japan). Quantitative real-time polymerase chain reaction studies All except 3 biopsy specimens (Cases 5, 8 and 13) were available for RT-PCR analyses. Total RNA was prepared from lung tissues, and complementary DNA was synthesized from total RNA using reverse transcriptase (TaKaRa Bio, Kusatsu, Japan). Quantitative RT-PCR was performed with the LightCycler96 system (Roche Diagnostics, Basel, Switzerland) using the following PCR conditions: 40 cycles 95 °C for 10 s, 52 °C for 10 s and 72 °C for 10 s. For ETBR, the PCR conditions were 40 cycles 95 °C for 20 s, 55 °C for 20 s and 72 °C for 20 s. The oligonucleotides used for PCR amplification are shown in Supplementary Document S2. Statistical analyses All values are presented as the mean ± standard deviation. When the data followed a normal distribution, comparisons between 2 groups were performed using the Student’s t-test. When the data did not follow a normal distribution, comparisons between 2 groups were performed using the Wilcoxon rank-sum test. All tests were 2-tailed, and statistical significance was assessed at the 5% α level. Research ethics board approval The research ethics committee of the University of Toyama approved this study. Written informed consent was obtained from the patients’ parents after the aim of these studies was explained to them. RESULTS Histopathological studies (DR=100μm) of each case are presented in Table 2. The mean (DR=100μm) values in patients with SV physiology were significantly greater than those in the control group (P = 0.0341, 12.6 ± 6.8 vs. 8.4 ± 1.0μm) (Fig. 1). Four patients (Cases 1, 3, 4 and 10) had severe medial hypertrophy of the pulmonary arteries (Fig. 2A). Their (DR=100μm)values amounted to nearly 20 μm. Three of these 4 patients (Cases 1, 3 and 4) failed or were not suitable for the Fontan operation, as described previously. The other patient (Case 10), who had asplenia syndrome, required release of a left pulmonary venous obstruction 10 months after the Fontan operation. A lung specimen was obtained from the patient’s left lung. The patient is doing well 2 years after the redo operation. Eight patients (Cases 5, 6, 7, 8, 9, 11, 12 and 13) with no or mild medial hypertrophy of the pulmonary arteries maintain good Fontan circulation (Fig 2B, 3C). There were no significant correlations between the (DR=100μm) values and any haemodynamic parameters. Figure 2: View largeDownload slide Histopathological findings of lung specimens. (A) Elastica van Gieson staining of the pulmonary artery in a patient who did not undergo a successful Fontan operation shows both severe intimal and medial hypertrophy (Case 1). (B) Elastica van Gieson staining of pulmonary arteries in a patient with a satisfactory clinical course showed slightly hypertrophic media (Case 7). (C) Masson’s trichrome staining of the pulmonary arteries in a normal control shows thin media. Figure 2: View largeDownload slide Histopathological findings of lung specimens. (A) Elastica van Gieson staining of the pulmonary artery in a patient who did not undergo a successful Fontan operation shows both severe intimal and medial hypertrophy (Case 1). (B) Elastica van Gieson staining of pulmonary arteries in a patient with a satisfactory clinical course showed slightly hypertrophic media (Case 7). (C) Masson’s trichrome staining of the pulmonary arteries in a normal control shows thin media. Figure 3: View largeDownload slide Endothelin-1 (green) and DAPI (blue) immunofluorescence staining of pulmonary arteries. (A) In a patient who did not undergo a successful Fontan operation, both the endothelium and media were strongly stained (Case 1). White arrowheads indicate the division between the media and the intima. (B) In a patient with a satisfactory clinical course, ET-1 was observed in the endothelium (Case 7). (C) In the normal control, the media and endothelium were barely stained. Figure 3: View largeDownload slide Endothelin-1 (green) and DAPI (blue) immunofluorescence staining of pulmonary arteries. (A) In a patient who did not undergo a successful Fontan operation, both the endothelium and media were strongly stained (Case 1). White arrowheads indicate the division between the media and the intima. (B) In a patient with a satisfactory clinical course, ET-1 was observed in the endothelium (Case 7). (C) In the normal control, the media and endothelium were barely stained. Immunohistochemical and immunofluorescence studies Three patients (Cases 1, 3, and 4) with severe medial hypertrophy of the pulmonary arteries showed a marked expression of ET-1 immunoreactivity in the endothelium and media of their pulmonary arteries (Fig. 3A). ET-1 immunoreactivity was mainly observed in the endothelium of pulmonary arteries in other patients and in those in the control group (Fig. 3B and C). Quantitative real-time polymerase chain reaction studies The results of RT-PCR analyses are shown in Table 2 and Fig. 4. Three patients who failed or were not suitable for the Fontan operation (Cases 1, 3 and 4) showed high levels of ET-1 expression, corresponding to the results of immunohistochemical and immunofluorescence studies. Two patients (Cases 11 and 12) showed high levels of ET-1. These 2 patients underwent redo common atrioventricular valve replacement for relative prosthetic valve obstruction because of somatic growth. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR expression, although these 2 cases maintain good Fontan circulation. There were no particular findings regarding the expression levels of ECE-1 and eNOS. There were no significant correlations between the duration of the Fontan circulation and the levels of ET-1, eNOS and ET receptors. Figure 4: View largeDownload slide Real-time polymerase chain reaction analyses of lung biopsies. (A) ET-1, (B) ETAR, (C) ETBR, (D) ECE-1 and (E) eNOS. Five patients (Cases 1, 3, 4, 11 and 12) showed high levels of ET-1. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR. Figure 4: View largeDownload slide Real-time polymerase chain reaction analyses of lung biopsies. (A) ET-1, (B) ETAR, (C) ETBR, (D) ECE-1 and (E) eNOS. Five patients (Cases 1, 3, 4, 11 and 12) showed high levels of ET-1. Two patients (Cases 7 and 9) showed high levels of ETAR and ETBR. DISCUSSION ET-1, a 21-amino acid peptide discovered in 1988 by Yanagisawa et al. [14], is one of the most potent endogenous vasoconstrictors and has mitogenic properties [15, 16]. ET-1 is strongly expressed in the lung, with ET-1 messenger RNA levels being at least 5-fold greater than in any other organ [17]. ET-1 in the pulmonary circulation can induce intense and protracted vasoconstriction of the pulmonary arteries and veins [18]. In addition to its effects on the pulmonary vascular tone, ET-1 also has a weak mitogenic effect on pulmonary vascular smooth muscle cells and stimulates matrix production by the vessel wall [19]. Although Hiramatsu et al. [5] demonstrated that ET-1 elevated PVR in patients with Fontan circulation, there were limited data about the relationship between ET-1 and the Fontan circulation. PVR is one of the most critical factors for maintaining the Fontan circulation and is dependent on various vasoactive factors, such as nitric oxide and prostacyclins. The pathophysiological role of ET-1 in PVR, in particular the potential involvement of ET-1 in patients with Fontan circulation, remains controversial. The aim of our study was thus to evaluate whether ET-1 plays an important role in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling in patients with SV physiology. We showed that patients who were unable to undergo a successful Fontan operation had both pulmonary arterial hypertrophy and the overexpression of ET-1 in the media of the pulmonary arteries using lung specimens. Maeda et al. [13] revealed that severe medial hypertrophy of the pulmonary arteries was correlated with a poor outcome of the Fontan operation. Indeed, our 3 patients (Cases 1, 3 and 4) with severe medial hypertrophy of the pulmonary arteries had poor outcomes (Fig. 2A). ET-1 immunoreactivity in these patients was observed in the endothelium and media (Fig. 3A). Thus, ET-1 may be associated with medial hypertrophy of the pulmonary arteries in the Fontan circulation. Conversely, patients without medial hypertrophy had good clinical courses (Fig. 2B), and ET-1 immunoreactivity was not observed in the media (Fig. 3B). Ishida et al. [10] also reported that the pulmonary arteries of the patients with failing Fontan circulation exhibited significant medial hypertrophy with proliferation of vascular smooth muscle cells and the expression of ET-1. ET-1 may play a role in medial hypertrophy of the pulmonary arteries in patients with Fontan circulation. We performed RT-PCR analyses to evaluate the quantity of the expression of ET-1. We revealed that the expression levels of ET-1 were increased in 5 patients (Cases 1, 3, 4, 11 and 12). Two of these patients (Cases 1 and 3) had poor early outcomes after the Fontan operation, despite normal PAP and PVR. One patient (Case 3) was not considered a candidate for Fontan completion because of pulmonary hypertension due to severe common atrioventricular regurgitation and numerous collateral arteries. These 3 patients had severe medial hypertrophy of the pulmonary arteries and marked expression of ET-1 immunoreactivity in the histopathological studies. However, vascular remodelling might be induced by ET-1, ET-1 would be one of the remodelling factors (cyanosis, common atrioventricular valve and pulmonary venous obstruction). Three of these 5 patients (Cases 3, 11 and 12) might have had an additional potential effect of post-capillary pulmonary hypertension. In our previous study of bosentan therapy, 2 of 8 patients showed an improvement in post-capillary pulmonary hypertension and underwent the Fontan operation successfully [11]. Bosentan therapy might be tried to treat post-capillary pulmonary hypertension, although the post-capillary pulmonary hypertension should be treated by surgical intervention if possible. Pulmonary arterial hypertrophy and the overexpression of ET-1 might be warnings of failing Fontan circulation, even though PAP and PVR were within normal limits. Because ET-1 also has a weak mitogenic effect on pulmonary vascular smooth muscle cells [19], this pathological status might disturb the Fontan circulation. The expressions of both ETAR and ETBR increased in 2 patients (Fig. 4B and C). These 2 patients (Cases 7 and 9) had satisfactory clinical courses. Ishida et al. [10] mentioned that the expression levels of ETAR and ETBR were significantly elevated in the pulmonary arteries of patients whose Fontan circulation failed. However, they did not study the endothelin system in patients who had good clinical courses after the Fontan operation. Recent pharmacological studies showed that ETAR and ETBR may form homodimers and/or heterodimers under pathological conditions [20, 21]. The Fontan circulation itself might be one of the pathological conditions. The expressions of ETAR and ETBR could increase in patients with Fontan circulation even if their circulation is good. Moreover, the endothelin systems might be complicated in patients with SV physiology. In the Fontan circulation, a non-pulsatile blood flow could create shear stress on the endothelium of the pulmonary system [22, 23]. As described previously, we reported that bosentan therapy reduced the PAP and PVR in patients who were unable to undergo a BDG or the Fontan operation because of high PAP and PVR [11]. Bosentan is an orally active, dual (ETAR and ETBR) endothelin receptor antagonist that can block both the detrimental effects of ET mediated via ETAR and the beneficial actions mediated via ETBR [24]. This study showed that ET-1 may play a role in promoting medial hypertrophy of the pulmonary arteries in patients with Fontan circulation. Our findings might suggest the validity of endothelin receptor antagonist therapy for patients with Fontan circulation. Limitations There are some limitations associated with this study. First, our study population was small. In the future, we should divide patients according to the type (Fontan or Glenn) of circulation. Second, we conducted immunohistochemical studies, which are not quantitative. In the RT-PCR studies, the lack of a comparison group of patients with BV physiology prevented us from presenting definitive evidence. Taking into consideration the multiple factors that may affect SV physiology, a group of patients with BV physiology would have been more suitable as a control group; however, such a group would have been difficult to incorporate into the study because few patients undergo lung biopsy without illness in infancy. CONCLUSIONS Severe medial hypertrophy of the pulmonary arteries was observed in patients who had poor outcomes. Marked expression of ET-1 was also observed in the endothelium and media of their pulmonary arteries. These pathological conditions may have some influence on the Fontan circulation. Thus, we concluded that there is an association between ET-1 and SV physiology in maintaining vasoconstriction of the pulmonary artery, which may subsequently promote vascular remodelling. 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Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

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Interactive CardioVascular and Thoracic SurgeryOxford University Press

Published: Mar 1, 2018

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